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Humbert, Lisa (2025) Fungal sex factors as novel antifungal compounds for control of plant disease. PhD thesis, University of Nottingham.

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Abstract

Global agriculture and wider food sectors face increasing pressure to produce sufficient, safe, high-quality, and affordable food and feed. This is due to the unprecedented combined challenges of climate change, a fast-growing human population, limited land and resources, regulatory pressures, evolution of antimicrobial resistance, societal demands, biodiversity decline, increasing greenhouse gas emissions and need for financial sustainability. The evolution of resistance by fungal plant pathogens to fungicides in agriculture underscores an urgent need for innovative crop protection solutions. Pyrenopeziza brassicae, the causative agent of light leaf spot disease in brassicas, is the most important fungal pathogen of oilseed rape in the UK. The disease is widespread across Northern Europe, Oceania, and Asia and has recently expanded into North America. The pathogen has well-described dispersal mechanisms, relying on asexual sporulation to facilitate local spread during the growing season, while sexual spore production enables long-distance transmission via ascospores in summer. Current reliance on broad-spectrum fungicides is increasingly ineffective due to widespread resistance in European strains, necessitating the development of novel targeted disease management solutions. Signalling molecules involved in fungal reproduction, such as sexual hormones, represent a promising new source of new tools for management of fungal pathogens, acting via developmental modification rather than toxic modes of actions. In P. brassicae, hormones referred to as sex factors (SF) produced during sexual reproduction have the ability to significantly suppress asexual sporulation of the species in laboratory conditions, while triggering the development of sterile sexual structures. Such sex factors are hypothesised to be responsible for the switch from asexual to sexual reproduction in the life cycle of the fungus. However, little is understood about the mode of action of sex factors. The overarching theme of this thesis is to explore the possible exploitation of sex factors for disease management, while deepening understanding of the role of SF in fungal sexual reproduction. Various parallel microbiological, genomic and biochemical approaches were used in studies.

The mode of action of SF was first explored at a morphological level. It was demonstrated that fungal cultures needed to reach a specific developmental stage (approximately 9-10 days post-inoculation) before they were able to respond to the sex factors. Subsequent scanning electron microscopy studies at the 10 dpi time point revealed the formation and expansion of a biofilm-like structure, trapping asexual spores and preventing their development and release. The limited number of asexual spores remaining exhibited structural deformation and had a 48 % decrease in their ability to germinate post SF treatment. The mode of action was then explored at a genetic level. Transcriptomic analysis of a single (unmated) P. brassicae isolate following SF treatment showed an overall downregulation of genes relating to asexual sporulation and upregulation of genes involved with sexual reproduction, including modification in expression of abaA, vosA, stuA, veA and velC, isp4, ste2, ste5, respectively. Taken with the morphological data, this suggests spore-suppressing activity of SF is regulated by both genetic mechanisms and the presence of a mechanical spore-trapping biofilm. Further analysis comparing differential gene expression at 10 and 15 days post SF treatment, combined with predicted protein structures, allowed the tentative identification of four candidate GPCRs as potential SF receptors. Ongoing in silico ligand-protein docking is aiming to narrow down to the receptor with the highest binding affinity to putative SF structures. Additionally, two genes encoding lipoxygenases with homology to Aspergillus ppoA genes were found in the genome of P. brassicae. These were considered possible candidates for biosynthetic genes involved in SF production. A protocol for CRISPR/Cas9 gene editing of P. brassicae was successfully developed and used to disrupt the ppoA2 genes in isolates of both mating types.

In parallel, attempts were made to purify and isolate the specific SF molecule(s). The sex factors were purified from crude lipid extracts of mated cultures of P. brassicae using a diverse range of analytical chemistry techniques. SF activity was located to two compounds that were characterised through LC-MS, MS/MS and NMR and assigned to tentative structures.

Finally, the application of sex factors as a possible disease management method was investigated both in planta and against global isolates of P. brassicae in vitro. European isolates from various origins and years strongly responded to SF treatment with an average of 97.5% decrease in sporulation. Overall, these findings deepen our understanding of fungal sexual reproduction and demonstrate that fungal sex hormones may offer more sustainable prospects for future crop protection.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Withall, David Dyer, Paul Birkett, Mike Brock, Matthias
Keywords:	Plant pathogens, Disease management, Fungal sex hormones, Pyrenopeziza brassicae
Subjects:	Q Science > QK Botany > QK710 Plant physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	81820
Depositing User:	Humbert, Lisa
Date Deposited:	10 Dec 2025 04:40
Last Modified:	10 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/81820

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